cDNA Cloning and Functional Expression of the Dolphin Retinal Rod Na−Ca+K Exchanger NCKX1:  Comparison with the Functionally Silent Bovine NCKX1

Abstract

cDNAs of human and bovine retinal rod Na+-Ca2++K+ exchanger (NCKX1) have previously been cloned, but potassium-dependent Na-Ca exchange activity upon heterologous expression has not been demonstrated. We have cloned NCKX1 cDNA from dolphin, examined function upon transfection in HEK293 cells, and compared the dolphin sequence encoded by the cDNA with those of human and bovine. The dolphin NCKX1 cDNA encodes 1013 amino acid residues. Comparison to bovine and human NCKX1 revealed strong conservation in the transmembrane domains (>95%), but relatively low conservation in the large extracellular ( approximately 50%) and cytosolic (<50%) domains. The dolphin cytosolic domain differs from the bovine sequence by the absence of a stretch of 114 amino acids. HEK293 cells transfected with dolphin NCKX1 cDNA showed K+-dependent Na-Ca exchange in >95% of the experiments, whereas transfection with bovine NCKX1 yielded no function. The bovine NCKX1 phenotype was imparted on dolphin NCKX1 when the dolphin cytosolic loop was replaced by that from bovine. Conversely, deletion of 114 amino acids from the bovine sequence to match the dolphin sequence resulted in a mutant bovine NCKX1 which performed K+-dependent Na-Ca exchange. These results suggest that domains within the large cytosolic loop of NCKX1 control functional activity when expressed in heterologous systems.

Molecular Cloning of a Sixth Member of the K+-dependent Na+/Ca2+ Exchanger Gene Family, NCKX6

“…we observe for NCKX6 (54). Transfected HEK-293 cells expressing bovine or dolphin NCKX1 molecules containing a region near the N-terminal end of the large cytoplasmic loop did not display Ca 2ϩ transport, whereas deletion of this region from either clone resulted in a functional exchanger, although protein expression from all these clones was similar.…”

Molecular Cloning of a Third Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family,NCKX3

“…The cloning of NCKX1 from rat eye (20) also revealed the presence of alternatively spliced isoforms in this species, which differ by the arrangement of four exons at the N terminus of the large intracellular loop. Interestingly, the equivalent region of bovine NCKX1, but apparently not the alternatively spliced region in rat NCKX1, is responsible for producing a functionally silent protein when expressed in heterologous systems (20,21).…”

A flagellar K + -dependent Na + /Ca 2+ exchanger keeps Ca 2+ low in sea urchin spermatozoa

“…Three NCKX subfamilies have been found in vertebrates, exchanging 1 K ϩ and 1 Ca 2ϩ (out) for 4 Na ϩ (in) (11). NCKX1 has been cloned from bovine (13), dolphin (14), chicken (15), rat (16), and human (17) retina rod cells. NCKX2 was originally identified in rat brain (18) and also retinal cone cells of chickens and humans (15).…”